Generally, the vascular endothelium and shear stress are critical determinants of hemostasis and platelet function in vivo, and yet, current diagnostic and monitoring devices do not fully incorporate endothelial function under flow in their assessment. Therefore, current diagnostic and monitoring devices can be unreliable and inaccurate. Furthermore, it is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust.
More specifically, mutual signaling between endothelium and activated platelets is widely recognized as critical for regulation of hemostasis and thrombotic disorders associated with various diseases, including atherosclerosis, sepsis, and diabetes. Yet, no practical diagnostic assays exist that can measure cross-talk between platelets and inflamed vessel walls in the presence of physiological shear. Over the last decade or so, multiple flow chambers and microfluidic devices that contain microchannels have been lined by living endothelium and exposed to flowing blood to study the basic science of thrombosis. While these devices have been very useful in advancing research, they have not been used in clinical settings due to the difficulty in maintaining living endothelial cells in them. Specifically, because it is extremely difficult to maintain the viability of living cell cultures for extended times in non-controlled settings, it is virtually impossible to rely on these assays. Therefore, the only microfluidic devices that are currently being deployed in clinical diagnostic settings are lined with collagen to mimic thrombus formation and platelet aggregation induced in response to vascular injury, and, thus, they fail to capture the physiological interplay between endothelial cells, platelets and fluid shear stress that is so relevant to hemostasis in inflammatory diseases.
Additionally, pulmonary microvascular thrombosis is a catastrophic condition amounting to a large number of patient deaths worldwide. Despite significant progress in understanding fundamental biology of lung hemostasis and thrombosis, it is still very difficult to predict response and study mechanism of action of potential drug candidates to humans. This is partly so because currently available in vitro assays do not recapitulate physiologically-relevant forces, such as shear stress, and animal models can be very complex allowing limited experimental manipulation, making it impossible to dissect and study intercellular signaling.
More specifically, pulmonary intravascular thrombosis and platelet activation initiating from, for example, acute lung injury (“ALI”), acute chest syndrome (“ACS”), pulmonary hypertension (“PH”), chronic obstructive pulmonary disease (“COPD”), and acute respiratory distress syndrome (“ARDS”), are causes of significantly high patient mortality and morbidity. Therefore, pulmonary intravascular thrombosis and platelet activation are also promising and emerging therapeutic targets to save and prolong patient life. Although epithelial injury, endothelial dysfunction, and in situ thrombotic lesions are observed often in human patients in chronic pulmonary diseases, animal models of pulmonary dysfunction are still unable to completely mimic the altered hemostasis and hemodynamic complexity of the lung. Importantly, animal models can be very complex and it may be impossible to study cell-cell interactions between multiple tissues independently of each other during blood clotting or drug administration. Based on this type of limitations, along with ethical barriers associated with animal models, it is desirable to advance in vitro disease models of pulmonary thrombosis that can mimic human organ-level functionality and complement or reduce reliance on animal studies, to enable more reliable basic research and make drug discovery more efficient.
In vitro, commercially available coagulation and platelet function technologies also have serious limitations due to the fact that they do not incorporate physiological tissue-tissue or cell-cell interactions, and relevant fluid dynamics of blood cells, which are key determinants of thrombosis. In research laboratories, dishes and transwell plates have been used for decades to culture cells and study basic biology, but these are static systems, highly non-physiological and cannot recapitulate tissue or organ-level functionality. For example, this type of systems cannot recapitulate blood flow or breathing of a lung.
To incorporate blood perfusion, parallel plate-flow chambers have been widely applied in the past three decades or so to measure thrombus formation and platelet adhesion kinetics. However, being macroscale devices, these chambers do not mimic small blood vessels, typically do not incorporate endothelium, and require large blood sample volumes for analysis.
More recently, microfluidic devices lined with human endothelial cells have shown that endothelial activation, platelet adhesion and fibrin formation in the presence of physiological shear can be somewhat visualized. However, these devices are also limited in studying organ-level pulmonary thrombosis, in part because they do not include the role of live epithelial cells, dynamic platelet-endothelial interactions (e.g., activation, aggregation, adhesion, translocation, and embolization) in the lumen that occur over large spatiotemporal scales, and often do not incorporate perfusion of whole blood.
Recently, microfluidic technology has been advanced to demonstrate an organ-level in vitro model of a lung and pulmonary edema, where alveolar epithelial and endothelial cells were co-cultured in two overlaying chambers, respectively. Fibrin formation in the alveolar chamber was analyzed in the presence of an inflammatory cytokine IL-2 and in the presence of flow and relevant cyclic stretch. However, this type of lung-on-a-chip model still lacks relevant functionality for mimicking relevant foundational conditions of pulmonary thrombosis. For example, the endothelial chamber only contains one side cultured with the cells and hence, it does not contain an endothelial lumen. Based on this limitation, the device is not appropriate for perfusing whole blood and for studying blood cell-endothelial interactions. In fact, other than a dilute suspension of neutrophils, none of the blood cells or platelets has been perfused or analyzed in this type of device, in its physiological concentration.
Another limitation of the long-on-a-chip model is that it uses non-primary epithelial cell lines, A549 or NCI-H441. Although this type of model mimics certain aspects of human lung function, it is not ideal in the context of mimicking physiologically-relevant hemostasis and thrombosis, as they are derived from tumors and, therefore, can potentially alter endothelial and platelet function.
Therefore, there is a continuing need for solving the above and other problems.